Cardiac myosin light chain kinase was purified from strips of ventricle and found to have a molecular weight of 70,000 in SDS-polyacrylamide gel electrophoresis. The enzyme requires both Ca2 ion and calmodulin for activity and has a specific activity of 1.2 micromoles Pi/mg kinase/min. Cardiac kinase can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, as determined by radioautography.